A DNA Revolution: Massively Parallel Sequencing

Scientists with Battelle Memorial Institute worked on a 19-month project to assess the feasibility of massively parallel sequencing as a forensic technique. Photo: Battelle

Three decades ago, DNA profiling was used for the first time in the United States, successfully tying a rapist to his crime. In Orange County, Florida in 1987, Tommy Lee Andrews was convicted of rape after tests matched his DNA from a blood sample with that of semen traces found in the rape victim.

Since then, DNA technology has held a prominence in the criminal justice system akin only to fingerprints. In that time, DNA has benefited from advanced technological innovations, such as the ability to use a wet vacuum collection system to pull microscopic trace DNA, or the advent of DNA software that can easily interpret mixtures once considered too complex to understand.

Still, like most things, DNA analysis does have its limitations. For example, using the industry-standard technique of capillary electrophoresis (CE), if a forensic analysist wants to examine the Y-chromosome, the X-chromosome and SNPs, each requires a separate amount of DNA—which, given the sample, is sometimes not possible—as well as a separate amplification and assay, increasing time, cost and efficiency.

That being said, CE has still proven to be exceptionally effective—as long as there is a suspect, or a match between the DNA sample and a national database. Without a suspect or match, the analysis stops and law enforcement is confronted with the beginnings of a cold case.

However, a new National Institute of Justice-ordered 19-month feasibility study suggests there’s a technology that may just overcome these limitations.

Massively parallel sequencing (MPS), also referred to as next-generation sequencing, is a high-throughput approach to DNA sequencing in which miniaturized, parallelized platforms are used for sequencing 1 million to 43 billion short reads per instrument run. The technical paradigm is built upon sequencing via spatially separated, clonally amplified DNA templates.

With MPS, forensic analysts are no longer limited to one type of marker at a time. Instead, an analyst can multiplex a number of them, say 20 autosomal STRs, 20 YSTRs, along with 100 SNPs standing at the end of the ancestral or phenotypic characteristics. Even if law enforcement doesn’t have a suspect or match, this extended ancestral or phenotypic information may provide some investigative or intelligence leads to build upon.

“[MPS] is not something that is going to be coming, it’s already here. It’s current,” said Richard Guerrieri, forensic research leader at Battelle, a non-profit R&D institute that manages multiple national laboratories.

The study

In 2015, the National Institute of Justice awarded Battelle a 19-month applied research project to evaluate the feasibility of MPS. With the overall goal to perform an objective assessment of the technology for forensic DNA testing labs, the project was split into two phases: 1) conduct the sequencing using commercially available products, and 2) evaluate and assess the fundamental elements of MPS analysis.

To do so, Battelle partnered with eight very different labs, in terms of size, mission and affiliation. This was done intentionally to evaluate if MPS could fit into all laboratories, regardless of capabilities. The eight labs included: Armed Forces DNA Identification Laboratory, Bureau of Alcohol, Tobacco and Firearms, California Department of Justice, Federal Bureau of Investigation, Harris County Institute of Forensic Sciences, National Institute of Standards and Technology, New York Office of the Chief Medical Examiner, and North Carolina State University.

There were four workflows to evaluate, one featuring Illumina products, two featuring Thermo Fisher Scientific products, and a final hybrid workflow featuring Promega, Illumina and Battelle products.

Each partner lab chose up to 12 non-probative casework samples to analyze, working on at least two of the four workflows. Some labs chose more classic forensic casework, such as bloodstains, while others chose specialized casework, like touch DNA samples or bones from WWII and the Korean War.

Overall, there was full concordance between MPS and reported CE data. All the partner labs and the different workflows were consistent and accurate, and there were no incorrect detections of DNA down to the level of single alleles. Sensitivity, accuracy, precision and reproducibility were all proven in the study, leading Guerrieri to conclude that “technical readiness was established, and the collective view is that MPS technology does have immediate value as a resource, especially for selected forensic applications.”

Different samples, more information

Beside concordance with established methods, one key takeaway from the project was the increased level of information available through sequencing technology.
For example, MPS proved especially effective on bone samples from the Korean War.

“Because the amplicons are much smaller in sequencing, you’re consequently able to accommodate samples that have been subjected to environmental abuse, resulting in degradation,” Guerrieri explained to Forensic Magazine. “DNA degrades from the outside in, and so fortunately if you have amplicons that are smaller, those regions are more protected from degradation.”

While the partner lab was successful using CE on the bone samples, MPS generated two to three times the amount of data.

In fact, Guerrieri sees the identification of human remains, missing persons and cold cases as areas where sequencing technology can really make a difference.

“Whether you couldn’t tease a profile out of that sample, or the suspect wasn’t in the national database, sequencing now has the capability and potential to solve some of those cases,” he said. “The possibility of bringing closure and resolution to something that has lingered for 25 years or longer, that to me is the big objective.”

In July 2016, Ohio’s Bureau of Criminal Investigation began the process of implementing MPS. While validation is still proceeding, the organization intends to go online, with services for the entire state of Ohio, no later than the end of March 2018. Thus far, the bureau has used the sequencing technology for missing persons cases and unidentified human remains. This is a step forward for the state, which had to previously outsource its missing persons cases to a lab based in Texas, causing time inefficiencies.

Additionally, one of the partner labs in the project, the Armed Forces DNA Identification Laboratory, has been using MPS technology for the last 12 months on selected cases dealing with bones, tissues, and missing persons.

Another area this “extra information” can prove useful is in dealing with mixtures, a hot topic in forensic science currently. While conventional methods for analyzing mixtures are limited to identifying the length of alleles, sequencing can show the construction of the bases that make up the units of alleles. Thus, even if two or three people in a mixture have the same length, MPS can tell them apart.

However, Guerrieri said there is more work that needs to be done with mixtures in general.

“There’s a lot of interest recently in trying to deconvolute mixtures, and the idea of using probabilistic modeling to further distinguish mixtures and the resulting information. That same approach can be successfully applied to this sequence-derived data, and I think it will actually be more effective,” he said.

For the project, Battelle scientists worked with a number of next generation sequencing systems, including Thermo Fisher Scientific’s Ion S5 (pictured). Photo: Battelle

The strategic roadmap

Like any new technology, there are still some kinks to work out as MPS moves forward. Forensic research leaders at Battelle said there are a few key technical areas in which further knowledge and advancement would be beneficial, including thresholds, how noise and remnant sequences of alleles may be related, and the effects of high-level multiplex amplification.

Other practical considerations include analysis cost, throughput and turnaround time. On a marker-to-marker basis, MPS is actually less expensive than CE, but that would predicate a lab using every marker, every time. In terms of time, MPS does not currently fit as the sole DNA method at a busy crime lab. For instance, it takes six to eight hours to obtain 20 CODIS markers from a DNA sample currently. MPS technology would produce 10 times the data, about 200 markers, but would take at least 24 hours to do so.

Data and practice standardization as well as policy are also elements that need to be considered in the future of MPS.

But perhaps the biggest issue going forward is the need for larger population databases, according to Guerrieri.

“Because you have more information, more alleles, more forms of genes that are being expressed, it would be a benefit to build the databases to be even larger for frequency determinations,” Guerrieri explained. “Now that you have more information, it would make sense to expand those databases on a technical level. In doing so, that gives you a better idea of how rare or how common some of those sequence variants can be.”

Battelle’s partner labs NIST and North Carolina State University have done work in this area already, and intend to share their population data with the community soon.

There are also several other forensic laboratories, not affiliated with the Battelle/NIJ project, that have teamed up and conducted their own internal validation of MPS, which they submitted to the FBI for approval in September 2017. The FBI is currently assessing the submissions for potential acceptance into the national database.

Once the process is completed, and the studies are indeed approved, Guerrieri believes it will be a turning point for sequencing.

“After the [Battelle] validation study and this second one get accepted by the FBI, I think there will be a lot of activity,” he said. “Now you can use sequencing technology to take advantage of the 15 million profiles in the national database.”